Stanford Researchers Find Loss of "Naive" T-Cells



Two studies by researchers at Stanford University have
questioned "conventional wisdom" on AIDS by finding a
relatively early, selective loss of "naive" CD4 and CD8 cells
in both HIV infected adults and children, respectively. In
view of these results, important beliefs about the
pathogenesis of HIV disease will need to be re-examined. AIDS
may be primarily a disease not of loss of CD4 cells, as in
generally believed, but of loss of naive cells of all types.
"This study forces us to re-evaluate all of the experiments
that have been done in the past 10 to 12 years on T-cell
function with cells from HIV patients," according to Mario
Roederer, the lead author of the adult study.

Both CD4 and CD8 cells can be subdivided into "naive" and
"memory" subsets. The ratio of naive to memory cells has now
been found to change greatly with HIV infection. For example,
about 50% of CD8 cells in uninfected adults are naive cells --
while less then 15% are naive in most HIV-infected adults.

Naive cells are cells which have never encountered the antigen
(foreign substance such as a protein produced by a bacterium
or virus) to which they are genetically able to respond. These
cells, therefore, are important because they allow the body to
develop immune responses to new challenges. Loss of naive
cells is expected to result in greater susceptibility to
certain opportunistic infections. In addition, this loss may
also erode the body's ability to control HIV, since many
different variants of HIV evolve in each patient, and without
enough naive cells the body cannot respond effectively to the
new ones.

The new findings are important for clinical trials, because
trials might be more accurate if volunteers were grouped
according to how many naive cells they have, as well as by how
many CD4 (T-helper) cells. And these findings may be
especially important for vaccine trials, since the lack of
naive cells will probably prevent the vaccines from working,
due to lack of immune response to the new antigen which a
vaccine provides.

Naive and memory cells also produce different cytokines. For
example, naive cells tend to produce IL-2, while memory cells
produce other cytokines such as IL-4, IL-10, and gamma
interferon. Therefore, measurements of the proportions of
different cytokines (the basis of theories about the switch
from a "Th1" to a "Th2" immune response) may be reflecting
differences in the number of naive and memory cells.

The study in adults tested blood samples from 266 HIV-infected
study volunteers, and from 44 uninfected healthy adults who
served as controls. The HIV-positive subjects were being
screened for a study of NAC (n-acetylcysteine), which was
seeking patients with a CD4 count under 500, and who were not
taking large amounts of antioxidants, vitamins, or minerals.

The parallel naive cell/memory cell study in children tested
19 HIV-infected children (who were recruited for a separate
study of glutathione levels) and 17 HIV-negative controls.

How the Measurements Were Made

Many different kinds of blood cells are best distinguished by
"markers" on their surface. These markers are large protein
molecules which are at the surface of the cell, and can
therefore interact with substances outside the cell. For
example, the CD4 (T-helper) and CD8 cells are distinguished by
the presence of CD4 or CD8 markers on their surfaces,
respectively. There are a number of other kinds of markers in
addition. Some kinds of cells have several different kinds of
markers on them. Usually each cell will have 10,000 to 100,000
copies of a marker; for example, CD4 or CD8 cells usually have
50,000 to 100,000 copies each.

These markers are detected by specially prepared antibodies
which are chemically attached to a substance which fluoresces
(glows) when exposed to a certain wavelength of light. Each
antibody is selected to attach only to a specific kind of
marker. Therefore, when one of the antibodies is mixed with
cells, many antibody molecules will attach to each cell which
expresses the target marker. These cells will then glow under
the special light, and they can be counted under a microscope.
This is how T-cell counts used to be done.

But several years ago a much better way of measuring T-cells
(and others cells distinguished by their markers) became
available. In this technology, called flow cytometry, a stream
of cells, one by one, rapidly moves in front of an ultraviolet
laser. When the laser hits the cell, the attached antibodies
glow, and this glow is measured and recorded in a computer;
the intensity of the glow indicates how many markers are on
that individual cell. Different antibodies can be engineered
to glow in different colors, allowing two or three different
markers to be measured simultaneously. Thousands of cells are
typically measured in one run; and the results can be plotted
on graph paper as clusters of dots, where each dot represents
one cell. By looking at these charts, it is possible to see if
certain populations of immune cells are missing or deficient,
resulting in specific immune defects. Two patients can be very
different in reality, even if their usual blood-count numbers
are the same.

Some previous studies have failed to find a selective loss of
naive T-cells. The Stanford researchers -- many of whom work
at the laboratory at Stanford where flow cytometry was
developed -- believe that earlier researchers failed to
measure naive cells correctly. Naive cells have the CD45RA
marker. But measuring that one alone may not be enough, since
two research groups previously determined that it is necessary
to measure three markers simultaneously (CD45RA, CD62L, and
either CD4 or CD8) to get an accurate count of naive cells.
This requires a "three color" flow cytometry machine, which
many though not all labs have.

Questions Remaining

Many important questions remain. For example, nobody knows why
the naive cells are selectively depleted. One theory is that
they are killed by HIV before they get out of the thymus
gland, where they develop. Damage to or deficiency of the
thymus might also contribute. But a completely different
possibility is that the immune system is overstimulated, and
abnormally causing naive cells to mature into memory cells.

Also, nobody knows what maintains the level of T-cells in
anyone's blood, whether or not they have HIV. More basic
research will be needed to answer these questions.

The new Stanford studies show the value of conducting basic
research in conjunction with clinical trials. Here, a basic
research study was piggybacked onto an ongoing trial of the
effect of NAC in persons with HIV; additional expense was
minimized, since their blood was being drawn and tested
anyway. But usually it is hard to include basic research in a
drug trial, since the pharmaceutical company sponsoring the
trial only want to know how well its drug works, and is
unlikely to pay for gathering knowledge which will help design
better treatments in the future.

References

Rabin RL, Roederer M, Maldonado Y, Petru A, Herzenberg LA, and
Herzenberg LA. Altered representation of naive and memory CD8
T cell subsets in HIV-infected children. JOURNAL OF CLINICAL
INVESTIGATION. May 1995; volume 95, pages 2054-2060

Roederer M, Dubs JG, Anderson MT, Raju PA, Herzenberg LA, and
Herzenberg LA. CD8 naive T cell counts decrease progressively
in HIV-infected adults. JOURNAL OF CLINICAL INVESTIGATION. May
1995; volume 95, pages 2061-2066.